Human PTTG polypeptide and method for producing it

ABSTRACT

Polypeptides are expressed by the pituitary-tumor-transforming-gene (PTTG), formerly known as pituitary-tumor-specific-gene (PTSG), and nucleic acids encode them. Examples are the human and rat PTTG proteins. The nucleic acids may be applied to the production of a recombinant protein, and to the detection of the presence of PTTG genes in different species. The nucleic acids may be operatively linked to a vector, optionally provided with control and expression sequences and/or being carried by a host cell. The nucleic acids may also be delivered to a mammal to compensate for the absence, or a defective expression, of endogenous protein. The nucleic acids, proteins, and antibodies are also employed in disgnostic assays, as well as, for example, in the production of anti-PTTG antibodies (protein), therapeutic compositions and other applications of the proteins and antibodies. Various kits utilize nucleic acids, polypeptides, and/or antibodies. A transgenic non-human mammal expresses PTTG.

[0001] This application claims the priority of the filing date of U.S.Provisional Application Serial No. 60/031,338, entitled NUCLEIC ACIDENCODING A FAMILY OF PITUITARY-TUMOR-SPECIFIC-GENES, AND PRODUCTSRELATED THERETO, by Shlomo Melmed and Lin Pei, filed Nov. 21, 1996.

[0002] This invention was made at least in part with United StatesGovernment support under Grant Number DK42742, awarded by the NationalInstitutes of Health. The Government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to nucleic acids and proteinsencoded thereby. Invention nucleic acids encode a novel family ofpituitary-tumor-specific-gene proteins. The invention also relates tomethods for making and using such nucleic acids and proteins.

[0005] 2. Description of the Background

[0006] Cancers and tumors are the second most prevalent cause of deathin the United States, causing 450,000 deaths per year. One in threeAmericans will develop cancer, and one in five will die of cancer(Scientific American Medicine, part 12, I, 1, section dated 1987). Whilesubstantial progress has been made in identifying some of the likelyenvironmental and hereditary causes of cancer, the statistics for thecancer death rate indicates a need for substantial improvement in thetherapy for cancer and related diseases and disorders.

[0007] A number of cancer genes, i.e., genes that have been implicatedin the etiology of cancer, have been identified in connection withhereditary forms of cancer and in a large number of well-studied tumorcells. Study of cancer genes have helped provide some understanding ofthe process of tumorigenesis. While a great deal more remains to belearned about cancer genes, the presently known cancer genes serve asuseful models for understanding tumorigenesis.

[0008] Cancer genes are broadly classified into “oncogenes” which, whenactivated, promote tumorigenesis, and “tumor suppressor genes” which,when damaged, fail to suppress tumorigenesis. While theseclassifications provide a useful method for conceptualizingtumorigenesis, it is also possible that a particular gene may playdiffering roles depending upon the particular allelic form of that gene,its regulatory elements, the genetic background and the tissueenvironment in which it is operating.

[0009] Tumor suppressor genes are genes that in their wild-type alleles,express proteins that suppress abnormal cellular proliferation. When thegene coding for a tumor suppressor protein is mutated or deleted, theresulting mutant protein or the complete lack of tumor suppressorprotein expression may fail to correctly regulate cellularproliferation, and abnormal cellular proliferation may take place,particularly if there is already existing damage to the cellularregulatory mechanism. A number of well-studied human tumors and tumorcell lines have been shown to have missing or nonfunctional tumorsuppressor genes. Examples of tumor suppression genes include, but arenot limited to the retinoblastoma susceptibility gen or RB gene, the p53gene, the deleted in colon carcinoma (DDC) gene and theneurofibromatosis type 1 (NF-1) tumor suppressor gene. Loss of functionor inactivation of tumor suppressor genes may play a central role in theinitiation and/or progression of a significant number of human cancers.

[0010] Anterior pituitary tumors are mostly benign hormone-secreting ornon-functioning adenomas arising from a monoclonal expansion of agenetically mutated cell. Pathogenesis of tumor formation in theanterior pituitary has been intensively studied. Mechanisms forpituitary tumorigenesis involve a multi-step cascade of recentlycharacterized molecular events. The most well characterized oncogene inpituitary tumors is gsp, a constitutively active Gasα resulting formactivating point mutations in this gene.

[0011] Gasα mutations occur in about 40% of GH-secreting tumors, andconstitutively activated CREB is also found in a subset of these tumors.Although the importance of GSα mutant proteins in the development ofgrowth-hormone secreting pituitary tumors is well established, onlyabout one third of these tumors contains these mutations, indicating thepresence of additional transforming events in pituitary tumorigenesis.Although point mutations of Ras oncogene, loss of heterozygosity (LOH)near the Rb locus on chromosome 13, and LOH on chromosome 11 have beenimplicated in some pituitary tumors, the mechanism that causes pituitarycell transformation remains largely unknown. Thus, there is a need inthe art for additional pituitary derived proteins that are associatedwith pituitary cell transformation.

SUMMARY OF THE INVENTION

[0012] The present invention relates to isolated, purifiedMammalian-pituitary-Transforming-Gene (PTTG) proteins, formerly namedMammalian Pituitary-Tumor-Specific-Gene (PTSG) proteins. The PTTGproteins of the invention and fragments thereof, are useful inbioassays, as immunogens for producing anti-PTTG antibodies, or intherapeutic compositions containing such proteins and/or antibodies.

[0013] This invention also relates to a transgenic non-human mammal thatexpresses PTTG protein.

[0014] The present invention also relates to isolated nucleic acidsencoding PTTG (PTSG) proteins of mammalian origin, such as human, rat,etc. The PTTG encoding nucleic acid is also provided in the form of avector carrying it, as hybridizing probes\primers, in host cellscarrying them, as anti-sense oligonucleotides, in DNA and RNA forms, andrelated compositions. The nucleic acid molecules described herein may beincorporated into expression systems known to those of skill in the art.The PTTG nucleic acids are useful as probes for assaying for thepresence and/or amount of a PTTG gene or mRNA transcript in a givensample. The nucleic acid molecules described herein, and oligonucleotidefragments thereof, are also useful as primers and/or templates in a PCRreaction for amplifying genes encoding PTTG proteins.

[0015] Antibodies that are immunoreactive with invention PTTG proteinsare also provided. These antibodies are useful in diagnostic assays todetermine levels of PTTG proteins present in a given sample, e.g.,tissue samples, biological fluids, Western blots, and the like. Theantibodies can also be used to purify PTTG proteins from crude cellextracts and the like. Moreover, these antibodies are consideredtherapeutically useful to counteract or supplement the biological effectof PTTGs in vivo.

[0016] Methods and diagnostic systems for determining the levels of PTTGprotein in various tissue samples are provided as well. These diagnosticmethods can be used for monitoring the level of therapeuticallyadministered PTTG protein or fragments thereof to facilitate themaintenance of therapeutically effective amounts. These diagnosticmethods can also be used to diagnose pathologic and physiologicaldisorders that result from abnormal levels of PTTG protein.

BRIEF DESCRIPTION OF THE FIGURE

[0017]FIG. 1 shows the effect of PTTG expression on cell proliferation.The cell growth rate is expressed as absorbance at 595 nM. The errorbars represent SEM (n=6). Three independent experiments were performed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0018] The present invention arose from a desire by the inventors toimprove over the prior art methods for detection of diseases orconditions associated with overproduction of, or abnormalities in, PTTGexpression. The inventors set out to isolate a PTTG gene in one speciesand then utilized probes based on the thus found gene sequence to probethe genomic DNA of other species, particularly human, and devise amethod for mutating the PTTG gene as well as a method for replacingdefective PTTG genes. They, in addition, conceived of a transgenicanimal for use as a model for the study of such diseases and conditionsin humans. This invention therefore provides isolated, purifiedmammalian pituitary-tumor-specific-gene (PTTG) proteins, polypeptides,and fragments thereof encoded by the nucleic acid of the invention. Asused herein, the phrase “PTTG” refers to a mammalian family of isolatedand/or substantially pure proteins, preferably human, that are able totransform cells in tissue culture, e.g., NIH 3T3 cells, and the like.The PTTG proteins of the invention have the ability to induce tumorformation in nude mice, e.g., when transfected into NIH 3T3 cells, andthe like. The PTTG proteins of the invention include naturally occurringallelic variants thereof encoded by mRNA generated by alternativesplicing of a primary transcript, and further include fragments whichretain at least one native biological activity, such as immunogenicity.

[0019] To identify genes specifically expressed in pituitary tumorcells, the inventors utilized GH-secreting and prolactin-secreting ratpituitary tumor cell lines. In this manner, they utilized them toeliminate the admixture of normal tissues present in surgically excisedhuman pituitary tumors and solid experimental rat tumors. Upon screeningabout 30% of expressed MRNA, a pituitary tumor transforming gene (PTTG)was identified and characterized. The sequence of PTTG revealed nohomology to any known sequences in the GenBank. The PTTG gene encodes aprotein of 199 amino acids that contains no characterized functionalmotif, which clearly indicates that PTTG is a novel protein.

[0020] The inventors showed the pituitary tumor specific expression ofPTTG by Northern blot analysis. Other than pituitary tumor cells, testistissue is the only normal non-tumor tissue to show PTTG expression.Interestingly, the PTTG messenger RNA in testis appears to be about 300bp shorter than that of the pituitary tumor, which indicates that thetestis messenger is a PTTG splice variant of the pituitary messenger..

[0021] The importance of PTTG in tumorigenesis is illustrated by itsability to transform 3T3 fibroblasts when overexpressed in these cells,as shown by morphological change and anchorage-independent growth ofPTTG transfectants in soft agar. This finding, moreover, is underscoredby the discovery that multiple tumor cell lines express abundant amountsof PTTG. Furthermore, nude mice injected with PTTG-expressing 3T3 cellsdeveloped large tumors within 3 weeks at all injection sites. These datashow that PTTG alone is capable of cellular transformation without therequirement of a complimentary oncogene, and that it is potentlytumorigenic in vivo. In general, full cell transformation requires twocomplementary oncogenes. See (Land et al, Nature, 304:696 (1983); Schwabet al., Nature, 316:160 (1985); Ruby et al., Nature 304:602 (1983). Insome cases, however, the overexpression of a single oncogene may besufficient to induce cellular transformation, as is the case in theRat-1 cell transformation by overexpression of the Ras gene alone. See,Reynolds, V L Oncogene, 1:323 (1987). In the present case, the inventorsfound that PTTG does not stimulate cell proliferation in culturedtransfected cells within 72 hours of assaying time. In fact, they foundthat PTTG unexpectedly inhibits all proliferation in culturedtransfected cells. This anti-proliferative effect is similar to thepotent inhibition of cell growth seen by Massague et al. with TGFβ. Oncethe cells are transformed, however, cell proliferation is accelerated,and rapid growth of tumors is seen in nude mice.

[0022] The PTSG proteins of this invention are polypeptide selectivelybound by anti-PTTG (anti-PTSG) antibody, the antibody preferably bindingto the human protein, including amino acid sequences SEQ ID NO:2, SEQ.ID No: 4, and fragments 5 to 50 amino acids long which bind to anti-PTTGantibody. The isolated PTSG proteins of the invention are generally freeof other cellular components and/or contaminants normally associatedwith a native in vivo environment, although they may have a certaincontent of these products, such as proteins, RNA, DNA andpolysaccharides..

[0023] The PTTG proteins are primarily, although not exclusively,expressed by pituitary tumor cells with expression detected in testis.The transcript in rat pituitary tumor cells is about 1.3 kb in sizewhile the transcript in testis is about 1 kb, as observed by a Northernblot assay. Splice variant cDNA-transcripts encoding a PTTG family ofproteins are clearly also contemplated by the present invention.

[0024] Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptide orproteins means that the DNA, RNA, polypeptide or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment. As a result ofthis human intervention, the recombinant DNAs, RNAs, polypeptide andproteins of the invention are useful in ways described herein that theDNAs, RNAs, polypeptide or proteins as they naturally occur are not.

[0025] As used herein, “mammalian” refers to the variety of species fromwhich the PTTG protein of the invention is derived from, e.g., human,rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine,feline, and the like. A preferred PTTG protein herein, is human PTTG.

[0026] Also part of this invention is the PTTG gene, which whendefective or present, is responsible for pituitary tumorigenesis. Asearch of GenBank and protein profile analysis (BLAST Program search ofdatabases of the national center for Biotechnology Information)indicated that PTTG shares no homology with known sequences, and itsencoded protein is highly hydrophilic, and contains no well recognizedfunctional motifs.

[0027] Presently preferred PTTG proteins of the invention include aminoacid sequences that are substantially the same as the amino acidsequence SEQ ID NO:2, SEQ. ID No: 4, and fragments thereof about 5 to 50amino acids long, as well as biologically active, modified formsthereof. Those of skill in the art will recognize that numerous residuesof the above-described sequences can be substituted with other,chemically, sterically and/or electronically similar residues withoutsubstantially altering the biological activity of the resulting receptorspecies. In addition, larger polypeptide sequences containingsubstantially the same sequence as SEQ ID NO:2 therein (e.g., splicevariants) are contemplated.

[0028] As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptide (or nucleic acids referred to hereinbefore)containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions, or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

[0029] The term “biologically active” or “functional”, when used hereinas a modifier of the PTTG protein(s) of this invention or polypeptidefragment thereof, refers to a polypeptide that exhibits at least one ofthe functional characteristics attributed to PTTG. For example, onebiological activity of PTTG is the ability to transform cells in vitro(e.g., NIH 3T3 cells, and the like). Yet another biological activity ofPTTG is the ability to induce tumor formation in nude mice (e.g., whentransfected into NIH 3T3 cells, and the like).

[0030] PTTG is also active as an immunogen for the production ofpolyclonal and monoclonal antibodies that bind selectively to PTTG.Thus, an invention nucleic acid encoding PTTG will encode a polypeptidespecifically recognized by an antibody that also specifically recognizesthe PTTG protein, preferably human, including amino acid sequences SEQID NO:2, SEQ. ID No: 4, and fragments thereof about 5 to 50 amino acidslong which bind to anti-PTTG antibody. Such activity may be assayed byany method known to those of skill in the art. For example, atest-polypeptide encoded by a PTTG cDNA may be used to produceantibodies, which are then assayed for their ability to bind to theprotein including the sequence set forth in SEQ ID NO:2. If the antibodybinds to the test-polypeptide and the protein including the sequence setforth in SEQ ID NO:2 with substantially the same affinity, then thepolypeptide possesses the requisite biological activity.

[0031] The PTTG proteins of the invention may be isolated by methodswell-known in the art, e.g., the recombinant expression systemsdescribed herein, precipitation, gel filtration, ion-exchange,reverse-phase and affinity chromatography, and the like. Otherwell-known methods are described in Deutscher et al., Guide to ProteinPurification: Methods in Enzymology Vol. 182, (Academic Press, (1990)),which is incorporated herein by reference. Alternatively, the isolatedpolypeptide of the present invention can be obtained using well-knownrecombinant methods as described, for example, in Sambrook et al.,supra., 1989).

[0032] An example of the means for preparing the inventionpolypeptide(s) is to express nucleic acids encoding the PTTG in asuitable host cell, such as a bacterial cell, a yeast cell, an amphibiancell (i.e., oocyte), or a mammalian cell, using methods well known inthe art, and recovering the expressed polypeptide, again usingwell-known methods. The PTTG polypeptide of the invention may beisolated directly from cells that have been transformed with expressionvectors as described herein. The invention polypeptide, biologicallyactive fragments, and functional equivalents thereof can also beproduced by chemical synthesis. For example, synthetic polypeptide canbe produced using Applied Biosystems, Inc. Model 430A or 431A automaticpeptide synthesizer (Foster City, Calif.) employing the chemistryprovided by the manufacturer.

[0033] Also encompassed by the term PTTG are polypeptide fragments orpolypeptide analogs thereof. The term “polypeptide analog” includes anypolypeptide having an amino acid residue sequence substantiallyidentical to a sequence specifically shown herein in which one or moreresidues have been conservatively substituted with a functionallysimilar residue and which displays the ability to mimic PTTG asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another. The phrase “conservativesubstitution” also includes the use of a chemically derivatized residuein place of a non-derivatized residue, provided that such polypeptidedisplays the requisite binding activity.

[0034] “Chemical derivative” refers to a subject polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include, for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptide of the present invention also include any polypeptide havingone or more additions and/or deletions of residues, relative to thesequence of a polypeptide whose sequence is shown herein, so long as therequisite activity is maintained.

[0035] The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified PTTG polypeptide, anactive fragment or polypeptide analog thereof, or a purified, matureprotein and active fragments thereof, alone or in combination with eachother. These polypeptide or proteins can be recombinantly derived,chemically synthesized or purified from native sources. As used herein,the term “acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents.

[0036] In accordance with another embodiment of the present invention,there are provided isolated nucleic acids, which encode the PTTG(pituitary-tumor-transforming-gene) proteins of the invention, andfragments thereof. The nucleic acid molecules described herein areuseful for producing invention proteins, when such nucleic acids areincorporated into a variety of protein expression systems known to thoseof skill in the art. In addition, such nucleic acid molecules orfragments thereof can be labeled with a readily detectable substituentand used as hybridization probes for assaying for the presence and/oramount of a PTTG gene or mRNA transcript in a given sample. The nucleicacid molecules described herein, and fragments thereof, are also usefulas primers and/or templates in a PCR reaction for amplifying genesencoding the invention protein described herein.

[0037] The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a PTTG protein. Onemeans of isolating a nucleic acid encoding an PTTG polypeptide is toprobe a mammalian genomic library with a natural or artificiallydesigned DNA probe using methods well known in the art. DNA probesderived from the PTTG gene are particularly useful for this purpose. DNAand cDNA molecules that encode PTTG polypeptide can be used to obtaincomplementary genomic DNA, cDNA or RNA from mammalian (e.g., human,mouse, rat, rabbit, pig, and the like), or other animal sources, or toisolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods described in more detail below. Examplesof nucleic acids are RNA, cDNA, or isolated genomic DNA encoding an PTTGpolypeptide. Such nucleic acids may include, but are not limited to,nucleic acids comprising SEQ ID NO: 1, alleles thereof, preferably atleast nucleotides 293-889 of SEQ ID NO: 1 or splice variant cDNAsequences thereof.

[0038] As used herein, the phrases “splice variant” or “alternativelyspliced”, when used to describe a particular nucleotide sequenceencoding an invention receptor, refers to a cDNA sequence that resultsfrom the well known eukaryotic RNA splicing process. The RNA splicingprocess involves the removal of introns and the joining of exons fromeukaryotic primary RNA transcripts to create mature RNA molecules of thecytoplasm. Methods of isolating splice variant nucleotide sequences arewell known in the art. For example, one of skill in the art may employnucleotide probes derived from the PTTG encoding DNA of SEQ ID NO: 1,SEQ. ID No: 3, alleles thereof, splice variants thereof or fragmentsthereof about 10 to 150 nucleotide long and their anti-sense nucleicacids to screen the cDNA or genomic library of the same or other speciesas described herein.

[0039] In one embodiment of the present invention, DNAs encoding thePTTG proteins of this invention comprise SEQ. ID NO: 1, SEQ. ID No: 3,alleles thereof, splice variants thereof and fragments thereof about 15to 150 nucleotide long and anti-sense nucleic acids thereof. In anotherembodiment of the present invention, DNA molecules encoding theinvention proteins comprise nucleotides 293-889 of SEQ ID NO: 1, allelesthereof, splice variants thereof and fragments thereof about 15 to 150nucleotide long. In yet another embodiment, the DNA comprisesnucleotides 292-899 of SEQ ID NO:3, alleles thereof, splice variantsthereof and fragments thereof about 10 to 150 nucleotides long.

[0040] As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in SEQ ID NO:2, or a larger amino acid sequenceincluding SEQ ID NO:2. In another embodiment, DNA having “substantiallythe same nucleotide sequence” as the reference nucleotide sequence hasat least 60% identity with respect to the reference nucleotide sequence.DNA having at least 70%, more preferably at least 90%, yet morepreferably at least 95%, identity to the reference nucleotide sequenceis preferred.

[0041] The present invention also encompasses nucleic acids which differfrom the nucleic acids shown in SEQ ID NO:1, but which have the samephenotype. Phenotypically similar nucleic acids are also referred to as“functionally equivalent nucleic acids”. As used herein, the phrase“functionally equivalent nucleic acids” encompasses nucleic acidscharacterized by slight and non-consequential sequence variations thatwill function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptide that are thesame as those disclosed herein or that have conservative amino acidvariations, or that encode larger polypeptide that include SEQ ID NO:2.For example, conservative variations include substitution of a non-polarresidue with another non-polar residue, or substitution of a chargedresidue with a similarly charged residue. These variations include thoserecognized by skilled artisans as those that do not substantially alterthe tertiary structure of the protein.

[0042] Further provided are nucleic acids encoding PTTG polypeptidesthat, by virtue of the degeneracy of the genetic code, do notnecessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. Preferred nucleic acids encoding the PTTGpolypeptide of the invention comprise nucleotides encoding SEQ ID NO:2,SEQ. ID No: 4, and fragments thereof about 5 to 50 amino acids long.Exemplary nucleic acids encoding a PTTG protein of the invention may beselected from the following.

[0043] (a) DNA encoding the amino acid sequence set forth in SEQ. IDNO:2,

[0044] (b) DNA that hybridizes to the DNA of (a) under moderatelystringent conditions, wherein said DNA encodes biologically active PTTG,or

[0045] (c) DNA degenerate with respect to either (a) or (b) above,

[0046] wherein said DNA encodes biologically active PTTG.

[0047] Hybridization refers to the binding of complementary strands ofnucleic acid (i.e., sense:antisense strands or probe:target-DNA) to eachother through hydrogen bonds, similar to the bonds that naturally occurin chromosomal DNA. Stringency levels used to hybridize a given probewith target-DNA can be readily varied by those of skill in the art.

[0048] The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

[0049] As used herein, the phrase “moderately stringent hybridization”refers to conditions that permit target-DNA to bind a complementarynucleic acid that has about 60% identity, preferably about 75% identity,more preferably about 85% identity to the target DNA; with greater thanabout 90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.

[0050] The phrase “high stringency hybridization” refers to conditionsthat permit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018 M NaCl at 65° C. (i.e., if a hybrid is notstable in 0.018 M NaCl at 65° C., it will not be stable under highstringency conditions, as contemplated herein). High stringencyconditions can be provided, for example, by hybridization in 50%formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42° C, followedby washing in 0.1×SSPE, and 0.1% SDS at 65° C.

[0051] The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5X Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989) are well known to those of skill in the art as are othersuitable hybridization buffers.

[0052] As used herein, the term “degenerate” refers to codons thatdiffer in at least one nucleotide from a reference nucleic acid, e.g.,SEQ ID NO: 1 or SEQ. ID No: 3, but encode the same amino acids as thereference nucleic acid. For example, codons specified by the triplets“UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respect to each othersince all four of these codons encode the amino acid serine.

[0053] Preferred nucleic acids encoding the invention polypeptide(s)hybridize under moderately stringent, preferably high stringency,conditions to substantially the entire sequence, or substantialportions, i.e., typically at least 15-30 nucleotide, of SEQ ID NO:1,SEQ. ID No: 3, although longer fragments are also contemplated.

[0054] Site-directed mutagenesis of any region of PTTG cDNA iscontemplated herein for the production of mutant PTTG cDNAs. Forexample, the Transformer Mutagenesis Kit (available from Clontech) canbe used to construct a variety of missense and/or nonsense mutations toPTTG cDNA.

[0055] The invention nucleic acids can be produced by a variety ofmethods well-known in the art, e.g., the methods described herein,employing PCR amplification using oligonucleotide primers from variousregions of SEQ ID NO:1, and the like.

[0056] In accordance with a further embodiment of the present invention,optionally labeled PTTG-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleic acid sequences encoding related novel mammalian PTTGproteins. Construction of mammalian cDNA and genomic libraries,preferably a human library, is well-known in the art. Screening of sucha cDNA or genomic library is initially carried out under low-stringencyconditions, which comprise a temperature of less than about 42° C., aformamide concentration of less than about 50%, and a moderate to lowsalt concentration.

[0057] Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5X standard saline citrate (SSC; 20×SSCcontains 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially thesame, i.e., similar, sequence as the coding region of the nucleic acidsof the invention, preferably as nucleotides 293-889 of SEQ ID NO: 1 areobtained.

[0058] As used herein, a nucleic acid “probe” is single-stranded DNA orRNA, or analogs thereof, that has a sequence of nucleotide that includesat least 14, preferably at least 20, more preferably at least 50,contiguous bases that are the same as (or the complement of) any 14 ormore contiguous bases set forth in any of SEQ ID NO: 1 or SEQ. ID No: 3.Preferred regions from which to construct probes include 5′ and/or 3′coding regions of SEQ ID NO: 1. In addition, the entire cDNA encodingregion of an invention PTTG protein, or the entire sequencecorresponding to SEQ ID NO: 1, may be used as a probe. Probes may belabeled by methods well-known in the art, as described hereinafter, andused in various diagnostic kits.

[0059] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

[0060] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturation to forma fluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

[0061] In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptide, and proteins, is well known in the art.For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:346 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

[0062] Also provided are antisense oligonucleotides having a sequencecapable of binding specifically with any portion of an mRNA that encodesPTTG polypeptide so as to prevent translation of the mRNA. The antisenseoligonucleotide may have a sequence capable of binding specifically withany portion of the sequence of the cDNA encoding PTTG polypeptide. Asused herein, the phrase “binding specifically” encompasses the abilityof a nucleic acid sequence to recognize a complementary nucleic acidsequence and to form double-helical segments therewith via the formationof hydrogen bonds between the complementary base pairs. An example of anantisense oligonucleotide is an antisense oligonucleotide comprisingchemical analogs of nucleotide.

[0063] Compositions comprising an amount of the antisenseoligonucleotide, described above, effective to reduce expression of PTTGpolypeptide by passing through a cell membrane and binding specificallywith mRNA encoding PTTG polypeptide so as to prevent translation and anacceptable hydrophobic carrier capable of passing through a cellmembrane are also provided herein. Suitable hydrophobic carriers aredescribed, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953;4,897,355, and the like. The acceptable hydrophobic carrier capable ofpassing through cell membranes may also comprise a structure which bindsto a receptor specific for a selected cell type and is thereby taken upby cells of the selected cell type. The structure may be part of aprotein known to bind to a cell-type specific receptor.

[0064] Antisense oligonucleotide compositions are useful to inhibittranslation of mRNA encoding invention polypeptide. Syntheticoligonucleotides, or other antisense chemical structures are designed tobind to mRNA encoding PTTG polypeptide and inhibit translation of mRNAand are useful as compositions to inhibit expression of PTTG associatedgenes in a tissue sample or in a subject.

[0065] In accordance with another embodiment of the invention, kits areprovided for detecting mutations, duplications, deletions,rearrangements or aneuploidies in the PTTG gene comprising at least oneinvention PTTG probe or antisense nucleotide.

[0066] The present invention provides means to modulate levels ofexpression of PTTG polypeptide by employing synthetic antisenseoligonucleotide compositions (hereinafter SAOC) which inhibittranslation of mRNA encoding these polypeptide. Syntheticoligonucleotides, or other antisense chemical structures designed torecognize and selectively bind to mRNA, are constructed to becomplementary to portions of the PTTG coding strand or nucleotidesequences shown in SEQ. ID No: 1 or SEQ. ID No: 3. The SAOC is designedto be stable in the blood stream for administration to a subject byinjection or by direct tumor site integration, or stable in laboratorycell culture conditions. The SAOC is designed to be capable of passingthrough the cell membrane in order to enter the cytoplasm of the cell byvirtue of physical and chemical properties of the SAOC which render itcapable of passing through cell membranes, for example, by designingsmall, hydrophobic SAOC chemical structures, or by virtue of specifictransport systems in the cell which recognize and transport the SAOCinto the cell. In addition, the SAOC can be designed for administrationonly to certain selected cell populations by targeting the SAOC to berecognized by specific cellular uptake mechanisms which bind and take upthe SAOC only within select cell populations.

[0067] For example, the SAOC may be designed to bind to a receptor foundonly in a certain cell type, as discussed supra. The SAOC is alsodesigned to recognize and selectively bind to target mRNA sequence,which may correspond to a sequence contained within the sequence shownin SEQ ID NO: 1. The SAOC is designed to inactivate target mRNA sequenceby either binding thereto and inducing degradation of the mRNA by, forexample, RNase I digestion, or inhibiting translation of mRNA targetsequence by interfering with the binding of translation-regulatingfactors or ribosomes, or inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups which either degrade orchemically modify the target mRNA. SAOCs have been shown to be capableof such properties when directed against mRNA targets (see Cohen et al.,TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), p.40;both incorporated herein by reference).

[0068] In accordance with yet another embodiment of the presentinvention, there is provided a method for the recombinant production ofthe PTTG protein(s) of the invention by expressing the above-describednucleic acid sequences in suitable host cells. Recombinant DNAexpression systems that are suitable to produce PTTG proteins describedherein are well-known in the art. For example, the above-describednucleotide sequences can be incorporated into vectors for furthermanipulation. As used herein, vector (or plasmid) refers to discreteelements that are used to introduce heterologous DNA into cells foreither expression or replication thereof.

[0069] Suitable expression vectors are well-known in the art, andinclude vectors capable of expressing DNA operatively linked to aregulatory sequence, such as a promoter region that is capable ofregulating expression of such DNA. Thus, an expression vector refers toa recombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. In addition, vectors may containappropriate packaging signals that enable the vector to be packaged by anumber of viral virions, e.g., retroviruses, herpes viruses,adenoviruses, resulting in the formation of a “viral vector”.

[0070] As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

[0071] As used herein, the term “operatively linked” refers to thefunctional relationship of DNA with regulatory and effector nucleotidesequences, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA.

[0072] As used herein, expression refers to the process by whichpolynucleic acids are transcribed into mRNA and translated intopeptides, polypeptide, or proteins. If the polynucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA.

[0073] Prokaryotic transformation vectors are well-known in the art andinclude pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla,Calif.), and the like. Other suitable vectors and promoters aredisclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989,the disclosure of which is incorporated herein by reference in itsentirety.

[0074] Other suitable vectors for transformation of E. coli cellsinclude the pET expression vectors (Novagen, see U.S. Pat. No.4,952,496), e.g., pET11a, which contains the T7 promoter, T7 terminator,the inducible E. coli lac operator, and the lac repressor gene; and pET12a-c, which contain the T7 promoter, T7 terminator, and the E. coliompT secretion signal. Another suitable vector is the pIN-IIIompA2 (seeDuffaud et al., Meth. in Enzymology, 153:492-507, 1987), which containsthe lpp promoter, the lacUV5 promoter operator, the ompA secretionsignal, and the lac repressor gene.

[0075] Exemplary, eukaryotic transformation vectors, include the clonedbovine papilloma virus genome, the cloned genomes of the murineretroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system(described by Mulligan and Berg, 1979, Nature Vol. 277:108-114) theOkayama-Berg cloning system (Mol. Cell Biol. Vol. 2:161-170, 1982), andthe expression cloning vector described by Genetics Institute (ScienceVol. 228:810-815, 1985), are available which provide substantialassurance of at least some expression of the protein of interest in thetransformed eukaryotic cell line.

[0076] Particularly preferred base vectors which contain regulatoryelements that can be linked to the invention PTTG-encoding DNAs fortransfection of mammalian cells are cytomegalovirus (CMV) promoter-basedvectors such as pcDNA1 (Invitrogen, San Diego, Calif.), MMTVpromoter-based vectors such as pMAMNeo (Clontech, Palo Alto, Calif.) andpMSG (Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors suchas pSVβ (Clontech, Palo Alto, Calif.).

[0077] In accordance with another embodiment of the present invention,there are provided “recombinant cells” containing the nucleic acidmolecules (i.e., DNA or mRNA) of the present invention. Methods oftransforming suitable host cells, preferably bacterial cells, and morepreferably E. coli cells, as well as methods applicable for culturingsaid cells containing a gene encoding a heterologous protein, aregenerally known in the art. See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York, USA (1989).

[0078] Exemplary methods of introducing (transducing) expression vectorscontaining invention nucleic acids into host cells to produce transducedrecombinant cells (i.e., cells containing recombinant heterologousnucleic acid) are well-known in the art (see, for review, Friedmann,1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, eachof which are incorporated herein by reference in their entirety).Exemplary methods of transduction include, e.g., infection employingviral vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764),calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and 4,634,665),dextran sulfate transfection, electroporation, lipofection (see, e.g.,U.S. Pat. Nos. 4,394,448 and 4,619,794), cytofection, particle beadbombardment, and the like. The heterologous nucleic acid can optionallyinclude sequences which allow for its extrachromosomal (i.e., episomal)maintenance, or the heterologous DNA can be caused to integrate into thegenome of the host (as an alternative means to ensure stable maintenancein the host).

[0079] Host organisms contemplated for use in the practice of thepresent invention include those organisms in which recombinantproduction of heterologous proteins has been carried out. Examples ofsuch host organisms include bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha andP. pastoris; see, e.g., U.S Pat. Nos. 4,882,279, 4,837,148, 4,929,555and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk⁻ cells),insect cells, and the like. Presently preferred host organisms arebacteria. The most preferred bacteria is E. coli.

[0080] In one embodiment, nucleic acids encoding the PTTG proteins ofthe invention may be delivered into mammalian cells, either in vivo orin vitro using suitable viral vectors well-known in the art, e.g.,retroviral vectors, adenovirus vectors, and the like. In addition, whereit is desirable to limit or reduce the in vivo expression of the PTTG ofthis invention, the introduction of the antisense strand of theinvention nucleic acid is contemplated.

[0081] Viral based systems provide the advantage of being able tointroduce relatively high levels of the heterologous nucleic acid into avariety of cells. Suitable viral vectors for introducing PTTG nucleicacid encoding a PTTG protein into mammalian cells (e.g., vascular tissuesegments) are well known in the art. These viral vectors include, forexample, Herpes simplex virus vectors (e.g., Geller et al., 1988,Science, 241:1667-1669), Vaccinia virus vectors (e.g., Piccini et al.,1987, Meth. in Enzymology, 153:545-563; Cytomegalovirus vectors(Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.78-84), Moloney murine leukemia virus vectors (Danos et al., 1980, PNAS,USA, 85:6469), adenovirus vectors (e.g., Logan et al., 1984, PNAS, USA,81:3655-3659; Jones et al., 1979, Cell, 17:683-689; Berkner, 1988,Biotechniques, 6:616-626; Cotten et al., 1992, PNAS, USA, 89:6094-6098;Graham et al., 1991, Meth. Mol. Biol., 7:109-127), adeno-associatedvirus vectors, retrovirus vectors, and the like. See, e.g., U.S. Pat.Nos. 4,405,712 and 4,650,764. Especially preferred viral vectors are theadenovirus and retroviral vectors.

[0082] For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., 1992, PNAS, USA, 89:6099-6103; Curiel et al., 1992, Hum.Gene Therapy, 3:147-154; Gao et al., 1993, Hum. Gene Ther., 4:14-24) areemployed to transduce mammalian cells with heterologous PTTG nucleicacid. Any of the plasmid expression vectors described herein may beemployed in a TfAdpl-DNA complex.

[0083] As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764).

[0084] Suitable retroviral vectors for use herein are described, forexample, in U.S. Pat. No. 5,252,479, and in WIPO publications WO92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829,incorporated herein by reference, which provide a description of methodsfor efficiently introducing nucleic acids into human cells using suchretroviral vectors. Other retroviral vectors include, for example, themouse mammary tumor virus vectors (e.g., Shackleford et al., 1988, PNAS,USA, 85:9655-9659), and the like.

[0085] In accordance with yet another embodiment of the presentinvention, there are provided anti-PTTG antibodies having specificreactivity with PTTG polypeptide of the present invention. Activefragments of antibodies are encompassed within the definition of“antibody”. Invention antibodies can be produced by methods known in theart using the PTTG polypeptide of the invention, proteins or portionsthereof as antigens. For example, polyclonal and monoclonal antibodiescan be produced by methods well known in the art, as described, forexample, in Harlow and Lane, Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory (1988)), which is incorporated herein byreference. The PTSG polypeptide of the invention may be utilized asimmunogens in generating such antibodies. Alternatively, syntheticpeptides can be prepared (using commercially available synthesizers) andused as immunogens. Amino acid sequences can be analyzed by methods wellknown in the art to determine whether they encode hydrophobic orhydrophilic domains of the corresponding polypeptide. Altered antibodiessuch as chimeric, humanized, CDR-grafted or bifunctional antibodies canalso be produced by methods well known in the art. Such antibodies canalso be produced by hybridoma, chemical synthesis or recombinant methodsdescribed, for example, in Sambrook et al., supra., and Harlow and Lane,supra. Both anti-peptide and anti-fusion protein antibodies can be used.(see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991);Ausubel et al., Current Protocols in Molecular Biology (John Wiley andSons, NY (1989) which are incorporated herein by reference).

[0086] Antibody so produced can be used, inter alia, in diagnosticmethods and systems to detect the level of PTTG protein present in amammalian, preferably human, body sample, such as tissue or vascularfluid. Such antibodies can also be used for the immunoaffinity oraffinity chromatography purification of the PTTG protein of theinvention. In addition, methods are contemplated herein for detectingthe presence of PTTG polypeptide either on the surface of a cell orwithin a cell (such as within the nucleus), which methods comprisecontacting the cell with an antibody that specifically binds to PTTGpolypeptide, under conditions permitting binding of the antibody to PTTGpolypeptide, detecting the presence of the antibody bound to PTTG, andthereby detecting the presence of invention polypeptide on the surfaceof, or within, the cell. With respect to the detection of suchpolypeptide, the antibodies can be used for in vitro diagnostic or invivo imaging methods.

[0087] Immunological procedures useful for in vitro detection of targetPTTG polypeptide in a sample include immunoassays that employ adetectable antibody. Such immunoassays include, for example, ELISA,Pandex microfluorimetric assay, agglutination assays, flow cytometry,serum diagnostic assays and immunohistochemical staining procedureswhich are well known in the art. An antibody can be made detectable byvarious means well known in the art. For example, a detectable markercan be directly or indirectly attached to the antibody. Useful markersinclude, for example, radionuclides, enzymes, fluorogens, chromogens andchemiluminescent labels.

[0088] The anti-PTTG antibodies of the invention modulate activity ofthe PTTG polypeptide in living animals, in humans, or in biologicaltissues or fluids isolated therefrom. Accordingly, compositionscomprising a carrier and an amount of an antibody having specificity forPTTG polypeptide effective to block naturally occurring ligands or otherPTTG-binding proteins from binding to invention PTTG polypeptide arecontemplated herein. For example, a monoclonal antibody directed to anepitope of PTTG polypeptide molecules present on the surface of a celland having an amino acid sequence substantially the same as an aminoacid sequence for a cell surface epitope of an PTTG polypeptideincluding the amino acid sequence shown in SEQ ID NO:2, SEQ. ID No: 4,and fragments thereof, may be useful for this purpose.

[0089] The present invention further provides transgenic non-humanmammals that are capable of expressing exogenous nucleic acids encodingPTTG polypeptide. As employed herein, the phrase “exogenous nucleicacid” refers to nucleic acid sequence which is not native to the host,or which is present in the host in other than its native environment(e.g., as part of a genetically engineered DNA construct). In additionto naturally occurring levels of PTTG, the PTTG proteins of thisinvention may either be overexpressed, underexpressed, or expressed inan inactive mutated form, such as in the well-known knock-outtransgenics, in transgenic mammals.

[0090] Also provided are transgenic non-human mammals capable ofexpressing nucleic acids encoding PTTG polypeptide so mutated as to beincapable of normal activity, i.e., do not express native PTTG. Thepresent invention also provides transgenic non-human mammals having agenome comprising antisense nucleic acids complementary to nucleic acidsencoding PTTG polypeptide, placed so as to be transcribed into antisensemRNA complementary to mRNA encoding PTTG polypeptide, which hybridizesto the mRNA and, thereby, reduces the translation thereof. The nucleicacid may additionally comprise an inducible promoter and/or tissuespecific regulatory elements, so that expression can be induced, orrestricted to specific cell types. Examples of nucleic acids are DNA orcDNA having a coding sequence substantially the same as the codingsequence shown in SEQ ID NO: 1. An example of a non-human transgenicmammal is a transgenic mouse.

[0091] Animal model systems which elucidate the physiological andbehavioral roles of PTTG polypeptide are also provided, and are producedby creating transgenic animals in which the expression of the PTTGpolypeptide is altered using a variety of techniques. Examples of suchtechniques include the insertion of normal or mutant versions of nucleicacids encoding an PTTG polypeptide by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal. (See, forexample, Hogan et al., Manipulating the Mouse Embryo: A LaboratoryManual (Cold Spring Harbor Laboratory, (1986)).

[0092] Also contemplated herein, is the use of homologous recombinationof mutant or normal versions of PTTG genes with the native gene locus intransgenic animals, to alter the regulation of expression or thestructure of PTTG polypeptide (see, Capecchi et al., Science 244:1288(1989); Zimmer et al., Nature 338:150 (1989); which are incorporatedherein by reference). Homologous recombination techniques are well knownin the art. Homologous recombination replaces the native (endogenous)gene with a recombinant or mutated gene to produce an animal that cannotexpress native (endogenous) protein but can express, for example, amutated protein which results in altered expression of PTTG polypeptide.

[0093] In contrast to homologous recombination, microinjection addsgenes to the host genome, without removing host genes. Microinjectioncan produce a transgenic animal that is capable of expressing bothendogenous and exogenous PTTG protein. Inducible promoters can be linkedto the coding region of nucleic acids to provide a means to regulateexpression of the transgene. Tissue specific regulatory elements can belinked to the coding region to permit tissue-specific expression of thetransgene. Transgenic animal model systems are useful for in vivoscreening of compounds for identification of specific ligands, i.e.,agonists and antagonists, which activate or inhibit protein responses.

[0094] Invention nucleic acids, oligonucleotides (including antisense),vectors containing same, transformed host cells, polypeptide andcombinations thereof, as well as antibodies of the present invention,can be used to screen compounds in vitro to determine whether a compoundfunctions as a potential agonist or antagonist to the PTTG polypeptideof the invention. These in vitro screening assays provide informationregarding the function and activity of the PTTG polypeptide of theinvention, which can lead to the identification and design of compoundsthat are capable of specific interaction with one or more types ofpolypeptide, peptides or proteins.

[0095] In accordance with still another embodiment of the presentinvention, there is provided a method for identifying compounds whichbind to PTTG polypeptide. The invention proteins may be employed in acompetitive binding assay. Such an assay can accommodate the rapidscreening of a large number of compounds to determine which compounds,if any, are capable of binding to PTTG proteins. Subsequently, moredetailed assays can be carried out with those compounds found to bind,to further determine whether such compounds act as modulators, agonistsor antagonists of invention proteins.

[0096] In another embodiment of the invention, there is provided abioassay for identifying compounds which modulate the activity of thePTTG polypeptide of the invention. According to this method, the PTTGpolypeptides of the invention are contacted with an “unknown” or testsubstance (in the presence of a reporter gene construct when antagonistactivity is tested), the activity of the polypeptide is monitoredsubsequent to the contact with the “unknown” or test substance, andthose substances which cause the reporter gene construct to be expressedare identified as functional ligands for PTTG polypeptide.

[0097] In accordance with another embodiment of the present invention,transformed host cells that recombinantly express the PTSG polypeptideof the invention may be contacted with a test compound, and themodulating effect(s) thereof can then be evaluated by comparing thePTTG-mediated response (e.g., via reporter gene expression) in thepresence and absence of test compound, or by comparing the response oftest cells or control cells (i.e., cells that do not express PTTGpolypeptide), to the presence of the compound.

[0098] As used herein, a compound or a signal that “modulates theactivity” of PTTG polypeptide of this invention refers to a compound ora signal that alters the activity of PTTG polypeptide so that theactivity of the invention PTTG polypeptide is different in the presenceof the compound or signal than in the absence of the compound or signal.In particular, such compounds or signals include agonists andantagonists. An agonist encompasses a compound or a signal thatactivates PTTG protein function. Alternatively, an antagonist includes acompound or signal that interferes with PTTG protein function.Typically, the effect of an antagonist is observed as a blocking ofagonist-induced protein activation. Antagonists include competitive andnon-competitive antagonists. A competitive antagonist (or competitiveblocker) interacts with or near the site specific for agonist binding. Anon-competitive antagonist or blocker inactivates the function of thepolypeptide by interacting with a site other than the agonistinteraction site.

[0099] As understood by those of skill in the art, assay methods foridentifying compounds that modulate PTTG activity generally requirecomparison to a control. One type of a “control” is a cell or culturethat is treated substantially the same as the test cell or test cultureexposed to the compound, with the distinction that the “control” cell orculture is not exposed to the compound. For example, in methods that usevoltage clamp electrophysiological procedures, the same cell can betested in the presence or absence of compound, by merely changing theexternal solution bathing the cell. Another type of “control” cell orculture may be a cell or culture that is identical to the transfectedcells, with the exception that the “control” cell or culture do notexpress native proteins. Accordingly, the response of the transfectedcell to compound is compared to the response (or lack thereof) of the“control” cell or culture to the same compound under the same reactionconditions.

[0100] In yet another embodiment of the present invention, theactivation of PTTG polypeptide can be modulated by contacting thepolypeptide with an effective amount of at least one compound identifiedby the above-described bioassays.

[0101] In accordance with another embodiment of the present invention,there are provided methods for diagnosing or detecting a pathologicalmass (such as, for example, an endocrine or non-endocrine tumor,atherosclerotic plaque, and the like), said method comprising detecting,in cells of a subject, a transcribed or mutant sequence including SEQ IDNO: 1.

[0102] In a particular embodiment, the invention diagnostic methodsdescribed herein are useful for differential diagnosis of malignantversus benign tumors in biopsy specimens, and the like. In anotherembodiment, the invention diagnostic methods described herein are usefulfor predicting tumor behavior and responsiveness to therapy.

[0103] In accordance with another embodiment of the present invention,there are provided methods for diagnosing pituitary tumors, said methodcomprising detecting, in pituitary-derived cells of a subject, atranscribed mRNA sequence including SEQ ID NO: 1.

[0104] In accordance with another embodiment of the present invention,there are provided diagnostic systems, preferably in kit form,comprising at least one invention nucleic acid in a suitable packagingmaterial. The diagnostic nucleic acids are derived from thePTTG-encoding nucleic acids described herein. In one embodiment, forexample, the diagnostic nucleic acids are derived from SEQ ID NO: 1.Invention diagnostic systems are useful for assaying for the presence orabsence of nucleic acid encoding PTTG in either genomic DNA or intranscribed nucleic acid (such as mRNA or cDNA) encoding PTTG in anytumors (e.g., pituitary, and the like) or diseased tissue. Inventiondiagnostic systems contemplated herein, may make use of well-knownpolymerase chain reaction (PCR) or RTPCR (reverse-transcriptase-PCR)methodologies.

[0105] A suitable diagnostic system includes at least one inventionnucleic acid, preferably two or more invention nucleic acids, as aseparately packaged chemical reagent(s) in an amount sufficient for atleast one assay. Instructions for use of the packaged reagent are alsotypically included. Those of skill in the art can readily incorporateinvention nucleic probes and/or primers into kit form in combinationwith appropriate buffers and solutions for the practice of the inventionmethods as described herein.

[0106] As employed herein, the phrase “packaging material” refers to oneor more physical structures used to house the contents of the kit, suchas invention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular sequence encoding PTTG including the nucleotidesequence set forth in SEQ ID NO: 1 or a mutant thereof, therebydiagnosing the presence of, or a predisposition for, a particularpathology (such as, for example, pituitary tumorigenesis, and the like).In addition, the packaging material contains instructions indicating howthe materials within the kit are employed both to detect a particularsequence and diagnose the presence of, or a predisposition for, aparticular pathology.

[0107] The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed. “Instructions for use” typically includea tangible expression describing the reagent concentration or at leastone assay method parameter, such as the relative amounts of reagent andsample to be admixed, maintenance time periods for reagent/sampleadmixtures, temperature, buffer conditions, and the like.

[0108] All U.S. patents and all publications mentioned herein areincorporated in their entirety by reference thereto. The invention willnow be described in greater detail by reference to the followingnon-limiting examples.

[0109] Unless otherwise stated, the present invention was performedusing standard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York, USA (1982); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987).

EXAMPLES Example 1: Isolation of PTTG cDNA

[0110] To clarify the molecular mechanisms involved in pituitarytumorigenesis, differential display PCR was used to identify mRNAsdifferentially expressed in pituitary tumor cells (see, e.g., Risingeret al., 1994, Molec. Carcinogenesis, 11:13-18; and Qu et al., 1996,Nature, 380:243-247). GC and GH₄ pituitary tumor cell lines (ATCC#CCL-82 and #CCL-82.1, respectively) and an osteogenic sarcoma cell lineUM108 (ATCC #CRL-1663) were grown in DMEM supplemented with 10% fetalbovine serum. Normal Sprague-Dawley rat pituitaries were freshlyexcised. Total RNA was extracted from tissue cultured cells andpituitary tissue using RNeasy™ kit (Qiagen) according to manufacturer'sinstructions. Trace DNA contamination in RNA preparations was removed byDNasel (GenHunter Corporation) digestion. cDNA was synthesized from 200ng total RNA using MMLV reverse transcriptase (GenHunter Corporation),and one of the three anchored primers (GenHunter Corporation). The cDNAgenerated was used in the PCR display.

[0111] Three downstream anchored primers AAGCT₁₁X (where X may be A, G,or C), were used in conjunction with 40 upstream arbitrary primers forPCR display. 120 primer pairs were used to screen mRNA expression inpituitary tumors versus normal pituitary. One tenth of the cDNAgenerated from the reverse transcriptase reaction was amplified usingAmpliTaq DNA polymerase (Perkin Elmer) in a total volume of 20 μlcontaining 10 mM Tris, pH 8,4, 50 nM KCl, 1.5 mM MgCl₂, 0.001% gelatin,2 μM dNTPs, 0.2 μM each primer and 1 μl [³⁵S]dATP. PCR cycles consistedof 30 seconds at 94° C., 2 minutes at 40° C., and 30 seconds at 72° C.for 40 cycles. The products were separated on 6% sequencing gels, anddried gels were exposed to Kodak film for 24 to 48 hours.

[0112] After development, DNA fragments amplified from pituitary tumorand normal pituitary were compared. Bands unique to pituitary tumor wereexcised from the gel, and DNA extracted by boiling in 100 μl water andprecipitated with ethanol in the presence of glycogen (GenHunterCorporation). DNA was reamplified using the original set of primers andthe same thermal cycling conditions except that the dNTP concentrationwas increased to 20 μM. Reaction products were run on 1% agarose gel andstained with ethidium bromide. Bands were excised from the gel, eluted(Qiagen), cloned in to TA vectors (Invitrogen) and sequenced usingsequenase (USB). Using 120 primer pairs in the above-described PCRassay, 11 DNA bands that appeared to be differentially expressed inpituitary tumor cells were identified. These bands were evaluatedfurther by Northern blot analysis, using the PCR products as probes.

[0113] For Northern blot analysis, 20 μg of total RNA were fractionatedon 1% agarose gel, blotted on to nylon membrane and hybridized withrandom primed probe using Quickhyb solutions (Stratagene). Afterwashing, membranes were exposed to Kodak films for 6 to 72 hours. As aresult of the Northern blot assay, pituitary tumor specific signals weredetected for 2 bands. DNA sequence analysis revealed that one sequencewas homologous with Insulin-induced growth response protein, while theanother 396 base pair fragment (amplified using 5′ AAGCTTTTTTTTTTTG 3′as the anchored primer and 5′ AAGCTTGCTGCTC 3′ as an arbitrary primer)showed no homology to known sequences in the GenBank. This 396 bpfragment detected a highly expressed mRNA of about 1.3 kb in pituitarytumor cells, but not in normal pituitary nor in osteogenic sarcomacells.

Example 2: Characterization of PTTG cDNA

[0114] To characterize this pituitary tumor-specific mRNA further, acDNA library was constructed using mRNA isolated from rat pituitarytumor cells. Poly A+RNA was isolated from pituitary tumor GH₄ cellsusing messenger RNA isolation kit (Stratagene) according tomanufacturer's instructions, and was used to construct a cDNA library inZAP Express vectors (Stratagene). The cDNA library was constructed usingZAP Express™ cDNA synthesis and Gigapack III gold cloning kit(Stratagene) following manufacturer's instructions. The library wasscreened using the 396 bp differentially displayed PCR product (clonedinto TA vector) as the probe. After tertiary screening, positive cloneswere excised by in vivo excision using helper phage. The resultingpBK-CMV phagemid containing the insert was identified by SouthernBlotting analysis. Unidirectional nested deletions were made into theDNA insert using EXOIII/Mung bean nuclease deletion kit (Stratagene)following manufacturer's instructions. Both strands of the insert DNAwere sequenced using Sequenase (USB).

[0115] Using the 396 bp PCR fragment described in Example 1 as a probe,a cDNA clone of 974 bp was isolated and characterized. This cDNA wasdesignated as pituitary-tumor-transforming gene (PTTG). The sequence ofPTTG contains an open reading frame for 199 amino acids (SEQ ID NO:2).The presence of an in-frame stop codon upstream of the predictedinitiation codon indicates that PTTG contains the complete ORF. Thenucleic acid and protein sequences are provided in Table 1 below. TABLE1 PTTG Nucleic Acid and Protein Sequences AATTCGGCAC GAGCCAACCTTGAGCATCTG ATCCTCTTGG CTTCTCCTTC CTATCGCTGA  60 GCTGGTAGGC TGGAGACAGTTGTTTGGGTG CCAACATCAA CAAACGATTT CTGTAGTTTA 120 GCGTTTATGA CCCTGGCGTGAAGATTTAAG GTCTGGATTA AGCCTGTTGA CTTCTCCAGC 180 TACTTCTAAA TTTTTGTGCATAGGTGCTCT GGTCTCTGTT GCTGCTTAGT TCTTCCAGCC 240 TTCCTCAATG CCAGTTTTATAATATGCAGG TCTCTCCCCT CAGTAATCCA GG ATG 295                                                          Met                                                            1 GCT ACTCTG ATC TTT GTT GAT AAG GAT AAC GAA GAG CCA GGC AGC CGT 343 Ala Thr LeuIle Phe Val Asp Lys Asp Asn Glu Glu Pro Gly Ser Arg              5                  10                  15 TTG GCA TCT AAGGAT GGA TTG AAG CTG GGC TCT GGT GTC AAA GCC TTA 391 Leu Ala Ser Lys AspGly Leu Lys Leu Gly Ser Gly Val Lys Ala Leu         20                  25                  30 GAT GGG AAA TTG CAGGTT TCA ACG CCA CGA GTC GGC AAA GTG TTC GGT 439 Asp Gly Lys Leu Gln ValSer Thr Pro Arg Val Gly Lys Val Phe Gly     35                  40                  45 GCC CCA GGC TTG CCT AAAGCC AGC AGO AAG GCT CTG GGA ACT GTC AAC 487 Ala Pro Gly Leu Pro Lys AlaSer Arg Lys Ala Leu Gly Thr Val Asn 50                  55                  60                  65 AGA GTTACT GAA AAG CCA GTG AAG AGT AGT AAA CCC CTG CAA TCG AAA 535 Arg Val ThrGlu Lys Pro Val Lys Ser Ser Lys Pro Leu Gln Ser Lys                 70                  75                  80 CAG CCG ACTCTG AGT GTG AAA AAG ATO ACC GAG AAG TCT ACT AAG ACA 583 Gln Pro Thr LeuSer Val Lys Lys Ile Thr Glu Lys Ser Thr Lys Thr             85                  90                  95 CAA GGC TCT GCTCCT GCT CCT GAT CAT GCC TAC CCA GAA ATA GAA AAG 631 Gln Gly Ser Ala ProAla Pro Asp Asp Ala Tyr Pro Glu Ile Glu Lys        100                 105                 110 TTC TTC CCC TTC CATCCT CTA GAT TTT GAG AGT TTT GAC CTG CCT GAA 679 Phe Phe Pro Phe Asp ProLeu Asp Phe Glu Ser Phe Asp Leu Pro Glu    115                 120                 125 GAG CAC CAG ATC TCA CTTCTC CCC TTG AAT GGA GTG CCT CTC ATG ATC 727 Glu His Gln Ile Ser Leu LeuPro Leu Asn Gly Val Pro Leu Met Ile130                 135                 140                 145 CTG AATGAA GAG AGO GGG CTT GAG AAG CTG CTG CAC CTG GAC CCC CCT 775 Leu Asn GluGlu Arg Gly Leu Glu Lys Leu Leu His Leu Asp Pro Pro                150                 155                 160 TCC CCT CTGCAG AAG CCC TTC CTA CCG TGG GAA TCT GAT CCG TTG CCG 823 Ser Pro Leu GlnLys Pro Phe Leu Pro Trp Glu Ser Asp Pro Leu Pro            165                 170                 175 TCT CCT CCC AGCGCC CTC TCC GOT CTG GAT GTT GAA TTG CCG CCT GTT 871 Ser Pro Pro Ser AlaLeu Ser Ala Leu Asp Val Glu Leu Pro Pro Val        180                 185                 190 TGT TAC GAT GCA GATATT TAAACGTCTT ACTCCTTTAT AGTTTATGTA 919 Cys Tyr Asp Ala Asp ILe    195                 200 AGTTGTATTA ATAAAGCATT TGTGTGTAAA AAAAAAAAAAAAAACTCGAG AGTAC 974

[0116] This was verified by demonstrating both in vitro transcriptionand in vitro translation of the gene product as described in Example 3.

Example 3: In vitro Transcription & Translation of PTTG

[0117] Sense and antisense PTTG mRNAs were in vitro transcribed using T3and T7 RNA polymerase (Stratagene), respectively. The excess templatewas removed by DNase I digestion. The in vitro transcribed mRNA wastranslated in rabbit reticular lysate (Stratagene). Reactions werecarried out at 30° C. for 60 minutes, in a total volume of 25 μlcontaining 3 μl in vitro transcribed RNA, 2 μl ³⁵S-Methionine (Dupond)and 20 μl lysate. Translation products were analyzed by SDS-PAGE (15%resolving gel and 5% stacking gel), and exposed to Kodak film for 16hours.

[0118] The results indicate that translation of in vitro transcribedPTTG sense mRNA results in a protein of approximately 25 KD on SDS-PAGE,whereas no protein was generated in either the reaction without addedmRNA or when PTTG antisense mRNA was utilized.

Example 4: Expression of PTTG mRNA

[0119] A search of GenBank and a protein profile analysis (using a BLASTProgram search of databases of the national center for BiotechnologyInformation) indicated that PTTG shares no homology with knownsequences, and its encoded protein is highly hydrophilic, and containsno well recognized functional motifs. The tissue expression patten ofPTTG mRNA was studied by Northern Blot analysis. A rat multiple tissueNorthern blot was purchased from Clontech. Approximately 2 μg of polyA+RNA per lane from eight different rat tissues (heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis) was run on adenaturing formaldehyde 1.2% agarose gel, transferred to nylon membraneand UV-cross linked. The membrane was first hybridized to the fulllength PTTG cDNA probe, and was stripped and rehybridized to a humanβ-actin cDNA control probe. Hybridization was performed at 60° C. forone hour in ExpressHyb hybridization solution (Clontech). Washing wastwice 15 minutes at room temperature in 2×SSC, 0.05% SDS, and twice 15minutes at 50° C. in 0.1% SSC, 0.1% SDS. Exposure time for PTTG probewas 24 hrs, and actin probe 2 hours.

[0120] The results of the Northern assay indicate that testis is theonly tissue, other than pituitary tumor cells, that expresses PTTG mRNA,and the testis expression level is much lower (2 μg polyA+mRNA, 24 hourexposure) than in pituitary tumor cells (20 μg total RNA, 6 hourexposure). Interestingly, the testicular transcript (about 1 Kb) isshorter than the transcript in pituitary tumors (1.3 Kb), indicatingthat the mRNA is differentially spliced in testis, and that the 1.3 Kbtranscript is specific for pituitary tumor cells.

Example 5: Over-Expression of PTTG by NIH 3T3 Fibroblast Cells

[0121] Since PTTG mRNA is over-expressed in pituitary tumor cells,whether this protein exerts an effect on cell proliferation andtransformation was determined. An eukaryotic expression vectorcontaining the entire coding region of PTTG was stably transfected intoNIH 3T3 fibroblasts.

[0122] The entire coding region of the PTTG was cloned in frame intopBK-CMV eukaryotic expression vector (Stratagene), and transfected intoNIH 3T3 cells by calcium precipitation. 48 hrs after transfection, cellswere diluted 1:10 and grown in selection medium containing 1 mg/ml G418for two weeks in when individual clones were isolated. Cell extractswere prepared from each colony and separated on 15% SDS-polyacrylamidegels, and blotted onto nylon membrane. A polyclonal antibody wasgenerated using the first 17 amino acids of PTTG as epitope (ResearchGenetics). The antibody was diluted 1:5000 and incubated with the abovemembrane at room temperature for 1 hour. After washing, the membrane wasincubated with horseradish peroxidase-labeled secondary antibody for onehour at room temperature. The hybridization signal was detected byenhanced chemiluminescence (ECL detection system, Amersham).

[0123] Expression levels of the PTTG were monitored by immunoblotanalysis using the above-described specific polyclonal antibody directedagainst the first 17 amino acids of the protein. Expression levels ofindividual clones varied, and clones that expressed higher proteinlevels were used for further analysis.

Example 6: Effect of PTTG Expression on Cell Proliferation

[0124] A nonradioactive cell proliferation assay was used to determinethe effect of PTTG protein over-expression on cell proliferation (see,e.g., Mosmann, T., 1983, J. Immunol. Meth., 65:55-63; and Carmichael etal., 1987, Cancer Res., 47:943-946). Cell proliferation was assayedusing CellTiter 96TM Non-radioactive cell proliferation Assay kit(Promega) according to the manufacturer's instructions. Five thousandcells were seeded in 96 well plates (6 wells for each clone in eachassay), and incubated at 37° C. for 24 to 72 hours. At each time point,15 μl of the Dye solution were added to each well, and incubated at 37°C. for 4 hours. One hundred μl of the solubilization/stop solution werethen added to each well. After one hour incubation, the contents of thewells were mixed, and absorbance at 595 nm was recorded using an ELISAreader. Absorbance at 595 nm correlates directly with the number ofcells in each well.

[0125] Three independent experiments were performed and the results areshown in FIG. 1. In FIG. 1, the cell growth rate is expressed asabsorbance at 595 nM. The error bars represent SEM (n=6). The results(FIG. 1) show that the growth rate of 3T3 cells expressing PTTG protein(assayed by cellular conversion of tetrazolium into formazan) wassuppressed 25 to 50% as compared with 3T3 cells expressing the pCMVvector alone, indicating that PTTG protein inhibits cell proliferation.

Example 7: PTTG Induction of Morphological Transformation and Soft-agarGrowth of NIH 3T3 Cells

[0126] The transforming property of PTTG protein was demonstrated by itsability to form foci in monolayer cultures and showanchorage-independent growth in soft agar, as shown in Table 2 below.TABLE 2 Colony Formation by NIH 3T3 Cells Transfected with PTTG cDNAConstructs Efficiency of Cell Growth in Colony formation line Soft Agarin Soft Agar (%) No DNA 0 0 Vector only 1.3 ± 0.7 0.013 PTTG 3  26 ± 4.60.26 PTTG 4 132 ± 26  1.32 PTTG 8  33 ± 6.0 0.33 PTTG 9 72 ± 13 0.72PTTG 10 92 ± 18 0.92

[0127] As primary pituitary cells are an admixture of multiple celltypes and they do not replicate in vitro, NIH 3T3 cells were employed.For the soft agar assay, 60 mm tissue culture plates were coated with 5ml soft-agar (20% 2XDMEM, 50% DMEM, 10% fetal bovine serum, 20% 2.5%agar, melted and combined at 45° C.). See, Schwab et al., 1985, Nature,316:160-162. 2 ml cells suspended in medium were then combined with 4 mlagar mixture, and 1.5 ml of this mixture added to each plate. The cellswere plated at a density of 10⁴ cells/dish and incubated for 14 daysbefore counting the number of colonies and photography.

[0128] The results indicate that NIH 3T3 parental cells and 3T3 cellstransfected with pCMV vector do not form colonies on soft agar, whereas3T3 cells transfected with PTTG form large colonies. In addition, focaltransformation is observed in cells over-expressing PTTG protein, butcells expressing pCMV vector without the PTTG insert showed similarmorphology to the parental 3T3 cells.

Example 8: Determination of In Vivo PTTG Tumorigenicity

[0129] To determine whether PTTG is tumorigenic in vivo,PTTG-transfected 3T3 cells were injected subcutaneously into athymicnude mice. 3×10⁵ cells of either PTTG or pCMV vector alone transfectedcells were resuspended in PBS and injected subcutaneously into nude mice(5 for each group). Tumors were excised from animals at the end of the3rd week and weighted. All injected animals developed large tumors (1-3grams) within 3 weeks. The results are shown in Table 3 below. No mouseinjected with vector only transfected cells developed tumors. TABLE 3 Invivo Tumorigenesis by NIH 3T3 Cells Transfected with PTTG cDNAExpression Vector Cell line Animals injected Tumor formation Vector only5 0/5 PTTG 4 5 5/5

[0130] These results clearly indicate that PTTG is a potent transforminggene in vivo.

Example 9: Human Carcinoma Cell Lines Express PTTG

[0131] The expression of PTTG in various human cell lines was studiedemploying a multiple human cancer cell line Northern blot (Clontech).The specific cell lines tested are shown in Table 4 below. TABLE 4 HumanCarcinoma Cell Lines Tested Cell Line PTTG Expression 1 PromyelocyticLeukemia HL-60 + 2 HeLa Cell S3 + 3 Chronic Myelogenous Leukemia K-562 +4 Lymphoblastic Leukemia MOLT-4 + 5 Burkitt's lymphoma Raji + 6Colorectal Adenocarcinoma SW 480 + 7 Lung Carcinoma A549 + 8 MelanomaG361 +

[0132] About 2 μg polyA RNA from each of the 8 cell lines indicated inTable 1 above were placed on each lane of a denaturing formaldehyde 1.2%agarose gel, separated by denaturing gel electrophoresis to ensureintactness, transferred to a charge-modified nylon membrane by Northernblotting, and fixed by UV irradiation. Lanes 1 to 8 contained RNA frompromyelocytic leukemia HL-60, HeLa cell line S3, human chronicmyelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt'slymphoma Raji, colorectal adenocarcinoma SW 480, lung carcinoma A549 andmelanoma G361, respectively. RNA size marker lines at 9.5, 7.5, 4.4,2.4, and 1.35 kb were indicated in ink on the left margin of the blot,and utilized as sizing standards, and a notch was cut out from the lowerleft hand corner of the membrane to provide orientation. Radiolabeledhuman β-actin cDNA was utilized as a control probe for matching ofdifferent batches of polyA RNAs. A single control band at 2.0 kb in alllanes spotted is confirmatory.

[0133] The blots were probed with the full length rat PTTG cDNA probe(SEQ. ID No: 1; 974 bp) at 60° C. for 1 hr. in ExpressHyb hybridizationsolution (Clontech) as described by Sambrook et al., the relevantsection of which reference is incorporated herein by reference. See,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Theblots were then washed twice for 15 min at room temperature in 2×SSC,0.05% SDS, and twice for 15 min at 50° C. in 0.1% SSC, 0.1% SDS. A moredetailed description of the remaining experimental procedures masy befound in Pei & Melmed, the relevant section of which is incorporatedherein by reference. See, Pei & Melmed, Endocrinology 4: 433441 (1997).

[0134] All cells tested by the Northern blot analysis as described aboveevidenced expression of human PTTG, including lymphoma, leukemia,melanoma and lung carcinomas, among others.

Example 10: Cloning of Human PTTG cDNA

[0135] A human fetal liver cDNA library (Clontech, Palo Alto, Calif.)was screened as described by Maniatis et al. using a radioactivelylabeled cDNA fragment of the entire rat PTTG coding region as a probe.See, Maniatis et al, Molecular cloning, Cold Spring Harbor Press, 1989.The cDNA inserts from positive clones were subcloned into plasmidpBluescript-SK (Stratagene, La Jolla, Calif.), and subjected to sequenceanalysis using Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio).The sequence of the nucleic acid is provided in Table 6 below. TABLE 6PTTG Nucleic Acid Sequences   1 ATGGCCGCGA GTTGTGGTTT AAACCAGGAGTGCCGCGCGT CCGTTCACCG  51 CGGCCTCAGA TGAATGCGGC TGTTAAGACC TGCAATAATCCAGAATG GCT 101 ACT CTG ATC TAT GTT GAT AAG GAA AAT GGA GAA CCA GGC ACC123 CGT GTG GTT GCT AAG GAT GGG CTG AAG CTG GGG TCT GGA CCT 185 TCA ATCAAA GCC TTA GAT GGG AGA TCT CAA GTT TCA ACA CCA 207 CGT TTT GGC AAA ACGTTC GAT QCC CCA CCA GCC TTA CCT AAA 249 GCT ACT AGA AAG GCT TTG GGA ACTGTC AAC AGA GCT ACA GAA 311 AAG TCT GTA AAG ACC AAG GGA CCC CTC AAA CAAAAA CAG CCA 350 CCA AGC TTT TCT GCC AAA AAG ATG ACT GAG AAG ACT GTT AAA392 GCA AAA AGC TCT GTT CCT GCC TCA CAT GAT GCC TAT CCA GAA 334 ATA GAAAAA TTC TTT CCC TTC AAT CCT CTA GAC TTT GAG AGT 376 TTT GAC CTG CCT GAAGAG CAC CAG ATT GCG CAC CTC CCC TTG 419 AGT GGA GTG CCT CTC ATG ATC CTTGAC GAG GAG AGA GAG CTT 560 GAA AAG CTG TTT CAG CTG GGC CCC CCT TCA CCTGTG AAG ATG 602 CCC TCT CCA CCA TGG GAA TCC AAT CTG TTG CAG TCT CCT TCA644 AGC ATT CTG TCG ACC CTG GAT GTT GAA TTG CCA CCT GTT TGC 686 TGT GACATA GAT ATT TAA 704     ATTTCTT AGTGCTTCAG AGTTTGTGTG TATTTGTATTAATAAAGCAT 751 TCTTTAACAG ATAAAAAAAA AAAAAAAAA (SEQ. ID No:3)

[0136] A complete open reading frame containing 606 bp was found in thepositive clones. The homology between the nucleotide sequences of theopen reading frame and the coding region of rat PTTG or PTTG (oldnomenclature) is 85%. The deduced amino acid sequence is shown in Table7 below. TABLE 7 PTTG Amino Acid Sequence   1 MATLIYVDKE NGEPGTRVVAKDGLKLGSGP SIKALDGRSQ VSTPRFGKTF  51 DAPPALPKAT RKALGTVNRA TEKSVKTKGPLKQKQPSFSA KKMTEKTVKA 101 KSSVPASDDA YPEIEKFFPF NPLDFESFDL PEEHQIAHLPLSGVPLMILD 151 EERELEKLFQ LGPPSPVKMP SPPWESNLLQ SPSSILSTLD VELPPVCCDI201 DI*

[0137] A comparison of the amino acid sequence the human PTTG translatedproduct of this open reading frame and that of the rat PTTG proteinreveals 77% identity and 89% homology. The cDNAs obtained from theseclones represents human homologies of rat PTTG. No other cDNA fragmentswith higher homology were detected from the library.

Example 11: Tissue Distribution of Human PTTG mRNA

[0138] Total RNA was prepared using Trizol Reagent (Gibco-BRL,Gaithersburg, Md.) from normal human pituitary glands (Zoion ResearchInc. Worcester, Mass.) and fresh human pituitary tumors collected atsurgery and frozen in liquid nitrogen. 20 mg total RNA were used for 1%agarose gel electrophoresis. RNA blots (Clontech, Palo Alto, Calif.)derived from normal adult and fetal tissues as well as from malignanttumor cell lines, were hybridized with radioactively labeled human cDNAfragment containing the complete coding region. The RNA isolated fromeach cell line was transferred onto a nylon membrane (Amersham,Arlington Heights, Ill.), and hybridized with radioactively labeledprobe at 55° C. overnight in 6×SSC, 2×Denhardt's solution, 0.25% SDS.The membranes were washed twice at room temperature for 15 minutes each,and then for 20 minutes at 60° C. in 0.5×SSC, 0.1% SDS, andautoradiographed. The autoradiography was carried out using KodakBIOMEX-MR film (Eastman Kodak, Rochester, N.Y.) with an intensifyingscreen. The blots were stripped by washing for 20 minutes in distilledwater at 95° C. for subsequent probing.

[0139] The results from the Northern blot analysis indicated that PTTGis expressed in liver, but not in brain, lung, and kidney of human fetaltissue. In addition, PTTG is strongly expressed in testis, modestlyexpressed in thymus, and weakly expressed in colon and small intestineof normasl human adult tissue. No expression was detected by Northernanalysis in brain, heart, liver, lung, muscle, ovary, placenta, kidney,and pancreas.

[0140] The expression of PTTG in several human carcinoma cell lines wasalso analyzed by Northern blots. In every carcinoma cells examined, PTTGwas found highly expressed. The human tumor cell lines tested are listedin Table 5 below. TABLE 5 Tested Human Tumor Cell Lines Promyelocyticleukemia HL-60 Epitheloid carcinoma HeLa cell S3 Chronic myelogenousleukemia K-562 Lymphoblastic leukemia MOLT-4 Burkitt's lymphoma RajiColorectal adenocarcinoma SW 480 Lung carcinoma A549 Melanoma G361Hepatocellular carcinoma Hep 3B Thyroid carcinoma TC-1 Breastadenocarcinoma MCF-7 Osteogenic sarcoma U2 OS Placenta choriocarcinomaJAR Choriocarcinoma JEG-3

Example 12: Human PTTG Expression in Normal Pituitary and PituitaryTumors

[0141] RT-PCR was performed as follows. 5 μg total RNA were treated with100 U RNase-free DNase I at room temperature for 15 minutes. DNase I wasinactivated by incubation at 65° C. for 15 minutes. The sample was thenused for reverse transcription using oligo-dT primer and SuperScript IIreverse transcriptase (Gibco-BRL, Gaithersburg, Md.). After reversetranscription, the sample was subjected to PCR amplification with PCRSuperMix (Gibco-BRL, Gaithersburg, Md.) using hPTTG-specific primers andhuman cyclophilin A-specific primers as an internal control.

[0142] Northern blot analysis indicated that the level of expression ofPTTG is quite low in normal pituitary as well as in pituitary tumors.Therefore, comparative RT-PCR was used to study the expression of PTTGquantitatively in normal pituitary and pituitary tumors. The results ofthis study showed that in most of pituitary tumors tested, includingnon-functioning tumors, GH-secreting tumors, and prolactinomas, theexpression level of PTTG was higher than that of normal pituitary.

Example 13: Stable Transfection of Human PTTG into NIH 3T3 Cells

[0143] The complete coding region of hPTTG cDNA was subcloned in readingframe into the mammalian expression vector pBK-CMV (Stratagene, LaJolla, Calif.), and transfected into NIH 3T3 fibroblast cells byLipofectamine (Gibco-BRL, Gaithersburg, Md.) according to manufacturer'sprotocol. 24 hours after transfection, the cells were serially dilutedand grown in selection medium containing 1 mg/ml G418 for 2 weeks.Individual clones were isolated and maintained in selection medium.Total RNA was isolated from hPTTG-transfected cell lines as well as fromcontrol cells in which blank vector pBK-CMV had been transfected.Northern blot was performed to confirm overexpression of hPTTG intransfected cell lines. These cell lines were used in subsequent cellproliferation assay as well as in vitro and in vivo transformationassay.

Example 14: Cell Proliferation Assay

[0144] A cell proliferation assay was performed using the CellTiter 96non-radioactive cell proliferation assay kit (Promega Medicine, Wis.)according to the manufacturer's protocol. 5,000 cells were seeded in96-well plates and incubated at 37° C. for 24-72 hours. Eight wells wereused for each clone in each assay. At each time point, 15 μl of dyesolution was added to each well and the cells were incubated at 37° C.for 4 hours. After incubation, 100 μl solubilization/stop solution wereadded to each well, and the plates incubated overnight at roomtemperature. The absorbance was determined at 595 nm using an ELISAplate reader.

[0145] Control and hPTTG-overexpressing NIH 3T3 cells were used toperform this assay. The results indicated that the growth of cellstransfected with the PTTG-expressing vector was suppressed by 30-45% ascompared with cells transfected with blank vector. These results clearlyshow that the PTTG protein inhibits cell proliferation.

Example 15: In Vitro and In Vivo Transformation Assay

[0146] (a) In Vitro Transformation Assay

[0147] Control and hPTTG-transfected cells were tested foranchorage-independent growth in soft agar. 3 ml of soft agar (20% of2×DMEM, 50% DMEM, 10% fetal bovine serum, and 20% of 2.5% agar, meltedand mixed at 45° C.) were added to 35 mm tissue dishes. 10,000 cellswere mixed with 1 ml soft agar and added to each dish, and iincubatedfor 2 weeks until colonies could be counted and photographed.

[0148] (b) In Vivo Transformation Assay

[0149] 5×10⁵ cells containing either a blank vector or hPTTG-expressingcells were injected into nude mice. The mice were sacrificed two weeksafter injection, and the tumors formed near the injection sitesexamined.

[0150] When the NIH 3T3 cells stably transfected with thePTTG-expressing vector were tested in an anchorage-independent growthassay, these cells caused large colony formation on soft agar,suggesting the transforming ability of PTTG protein.

[0151] When the NIH 3T3 cells were injected into nude mice, they causedin vivo tumor formation within 2 weeks after injection. These dataindicate that human PTTG, as its rat homologue, is a potent transforminggene.

SUMMARY OF SEQUENCES

[0152] SEQ ID NO:1 is the nucleic acid sequence (and the deduced aminoacid sequence) of cDNA encoding the rat PTTG protein of the presentinvention.

[0153] SEQ ID NO:2 is the deduced amino acid sequence of the rat PTTGprotein of the present invention.

[0154] SEQ. ID No:3 is the nucleic acid sequence of cDNA encoding ahuman PTTG protein of this invention.

[0155] SEQ. ID No:4 is the deduced amino acid sequence of a human PTTGprotein of this invention.

[0156] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it should be understood thatmodifications and variations to the embodiments and exemplary disclosureprovided, are within the spirit and scope of the invention as describedand claimed in this patent.

1 4 1 974 DNA Rattus rattus 1 aattcggcac gagccaacct tgagcatctgatcctcttgg cttctccttc ctatcgctga 60 gctggtaggc tggagacagt tgtttgggtgccaacatcaa caaacgattt ctgtagttta 120 gcgtttatga ccctggcgtg aagatttaaggtctggatta agcctgttga cttctccagc 180 tacttctaaa tttttgtgca taggtgctctggtctctgtt gctgcttagt tcttccagcc 240 ttcctcaatg ccagttttat aatatgcaggtctctcccct cagtaatcca ggatggctac 300 tctgatcttt gttgataagg ataacgaagagccaggcagc cgtttggcat ctaaggatgg 360 attgaagctg ggctctggtg tcaaagccttagatgggaaa ttgcaggttt caacgccacg 420 agtcggcaaa gtgttcggtg ccccaggcttgcctaaagcc agcaggaagg ctctgggaac 480 tgtcaacaga gttactgaaa agccagtgaagagtagtaaa cccctgcaat cgaaacagcc 540 gactctgagt gtgaaaaaga tcaccgagaagtctactaag acacaaggct ctgctcctgc 600 tcctgatgat gcctacccag aaatagaaaagttcttcccc ttcgatcctc tagattttga 660 gagttttgac ctgcctgaag agcaccagatctcacttctc cccttgaatg gagtgcctct 720 catgatcctg aatgaagaga gggggcttgagaagctgctg cacctggacc ccccttcccc 780 tctgcagaag cccttcctac cgtgggaatctgatccgttg ccgtctcctc ccagcgccct 840 ctccgctctg gatgttgaat tgccgcctgtttgttacgat gcagatattt aaacgtctta 900 ctcctttata gtttatgtaa gttgtattaataaagcattt gtgtgtaaaa aaaaaaaaaa 960 aaactcgaga gtac 974 2 199 PRTRattus rattus 2 Met Ala Thr Leu Ile Phe Val Asp Lys Asp Asn Glu Glu ProGly Ser 1 5 10 15 Arg Leu Ala Ser Lys Asp Gly Leu Lys Leu Gly Ser GlyVal Lys Ala 20 25 30 Leu Asp Gly Lys Leu Gln Val Ser Thr Pro Arg Val GlyLys Val Phe 35 40 45 Gly Ala Pro Gly Leu Pro Lys Ala Ser Arg Lys Ala LeuGly Thr Val 50 55 60 Asn Arg Val Thr Glu Lys Pro Val Lys Ser Ser Lys ProLeu Gln Ser 65 70 75 80 Lys Gln Pro Thr Leu Ser Val Lys Lys Ile Thr GluLys Ser Thr Lys 85 90 95 Thr Gln Gly Ser Ala Pro Ala Pro Asp Asp Ala TyrPro Glu Ile Glu 100 105 110 Lys Phe Phe Pro Phe Asp Pro Leu Asp Phe GluSer Phe Asp Leu Pro 115 120 125 Glu Glu His Gln Ile Ser Leu Leu Pro LeuAsn Gly Val Pro Leu Met 130 135 140 Ile Leu Asn Glu Glu Arg Gly Leu GluLys Leu Leu His Leu Asp Pro 145 150 155 160 Pro Ser Pro Leu Gln Lys ProPhe Leu Pro Trp Glu Ser Asp Pro Leu 165 170 175 Pro Ser Pro Pro Ser AlaLeu Ser Ala Leu Asp Val Glu Leu Pro Pro 180 185 190 Val Cys Tyr Asp AlaAsp Ile 195 3 779 DNA Homo sapiens 3 atggccgcga gttgtggttt aaaccaggagtgccgcgcgt ccgttcaccg cggcctcaga 60 tgaatgcggc tgttaagacc tgcaataatccagaatggct actctgatct atgttgataa 120 ggaaaatgga gaaccaggca cccgtgtggttgctaaggat gggctgaagc tggggtctgg 180 accttcaatc aaagccttag atgggagatctcaagtttca acaccacgtt ttggcaaaac 240 gttcgatgcc ccaccagcct tacctaaagctactagaaag gctttgggaa ctgtcaacag 300 agctacagaa aagtctgtaa agaccaagggacccctcaaa caaaaacagc caagcttttc 360 tgccaaaaag atgactgaga agactgttaaagcaaaaagc tctgttcctg cctcagatga 420 tgcctatcca gaaatagaaa aattctttcccttcaatcct ctagactttg agagttttga 480 cctgcctgaa gagcaccaga ttgcgcacctccccttgagt ggagtgcctc tcatgatcct 540 tgacgaggag agagagcttg aaaagctgtttcagctgggc cccccttcac ctgtgaagat 600 gccctctcca ccatgggaat ccaatctgttgcagtctcct tcaagcattc tgtcgaccct 660 ggatgttgaa ttgccacctg tttgctgtgacatagatatt taaatttctt agtgcttcag 720 agtttgtgtg tatttgtatt aataaagcattctttaacag ataaaaaaaa aaaaaaaaa 779 4 202 PRT Homo sapiens 4 Met Ala ThrLeu Ile Tyr Val Asp Lys Glu Asn Gly Glu Pro Gly Thr 1 5 10 15 Arg ValVal Ala Lys Asp Gly Leu Lys Leu Gly Ser Gly Pro Ser Ile 20 25 30 Lys AlaLeu Asp Gly Arg Ser Gln Val Ser Thr Pro Arg Phe Gly Lys 35 40 45 Thr PheAsp Ala Pro Pro Ala Leu Pro Lys Ala Thr Arg Lys Ala Leu 50 55 60 Gly ThrVal Asn Arg Ala Thr Glu Lys Ser Val Lys Thr Lys Gly Pro 65 70 75 80 LeuLys Gln Lys Gln Pro Ser Phe Ser Ala Lys Lys Met Thr Glu Lys 85 90 95 ThrVal Lys Ala Lys Ser Ser Val Pro Ala Ser Asp Asp Ala Tyr Pro 100 105 110Glu Ile Glu Lys Phe Phe Pro Phe Asn Pro Leu Asp Phe Glu Ser Phe 115 120125 Asp Leu Pro Glu Glu His Gln Ile Ala His Leu Pro Leu Ser Gly Val 130135 140 Pro Leu Met Ile Leu Asp Glu Glu Arg Glu Leu Glu Lys Leu Phe Gln145 150 155 160 Leu Gly Pro Pro Ser Pro Val Lys Met Pro Ser Pro Pro TrpGlu Ser 165 170 175 Asn Leu Leu Gln Ser Pro Ser Ser Ile Leu Ser Thr LeuAsp Val Glu 180 185 190 Leu Pro Pro Val Cys Cys Asp Ile Asp Ile 195 200

1. A purified, isolated pituitary-tumor-transforming-gene (PTTG)polypeptide expressed by pituitary tumor cells, which binds anti-PTTGantibody.
 2. The polypeptide of claim 1, comprising the amino acidsequence encoded by the coding regions of SEQ. ID No: 1, of SEQ. ID No:3, and of fragments thereof 5 to 50 amino acids long which bindanti-PTTG antibody.
 3. The polypeptide of claim 2, comprising the aminoacid sequence encoded by the coding region of SEQ. ID No:
 1. 4. Thepolypeptide of claim 3, comprising nucleotides 292 to 899 of SEQ. IDNo:
 1. 5. The polypeptide of claim 2, comprising fragments 5 to 50 aminoacids long of the amino acid sequence encoded by the coding region ofSEQ. ID No:
 1. 6. The polypeptide of claim 2, comprising the amino acidsequence encoded by the coding sequence of SEQ ID No:
 3. 7. Thepolypeptide of claim 6, comprising the amino acid sequence encoded bynucleotides 292 to 899 of SEQ. ID No:
 3. 8. The polypeptide of claim 6,comprising fragments 5 to 50 amino acids long of the amino acid sequenceencoded by the coding region of SEQ. ID No:
 3. 9. The polypeptide ofclaim 2, comprising an amino acid sequence selected from the groupconsisting of SEQ. ID No: 2, SEQ. ID No: 4 and fragments thereof 5 to 50amino acids long which bind anti-PTTG antibody.
 10. The polypeptide ofclaim 9, comprising SEQ. ID No:
 2. 11. The polypeptide of claim 9,comprising SEQ ID NO:
 4. 12. The polypeptide of claim 9, comprisingfragments of SEQ. ID No: 2 5 to 50 amino acids long which bind anti-PTTGantibody.
 13. The polypeptide of claim 9, comprising fragments of SEQ.ID No: 4 5 to 50 amino acids long which bind anti-PTTG antibody.
 14. Thepolypeptide of claim 1, which is of mammalian origin.
 15. Thepolypeptide of claim 14, wherein its origin is selected from the groupconsisting of human, rat, mouse, porcine, ovine, canine, human andbovine origins.
 16. A composition, comprising the polypeptide of claim1, and a carrier.
 17. An isolated, purified polynucleotide, comprising anucleic acid encoding the polypeptide of claim
 1. 18. The polynucleotideof claim 17, comprising a nucleic acid selected from the groupconsisting of SEQ. ID No: 1, SEQ. ID No: 3, and fragments thereof 10 to150 nucleotide long.
 19. The polynucleotide of claim 17, comprising anucleic acid selected from the group consisting of (a) a nucleic acidselected from the group consisting of those encoding SEQ ID NO:2, SEQ.ID No: 4, and fragments thereof 5 to 50 amino acids long bindinganti-PTTG antibody; (b) splice variant sequences thereof; (c)probe/primer fragments thereof; oand (c) nucleic acid that hybridizes tothe nucleic acid of (a) under moderately stringent conditions.
 20. Thepolynucleotide of claim 17, comprising a nucleic acid selected from thegroup consisting of those that hybridize under high stringencyconditions to the PTTG coding region of SEQ. ID No:1.
 21. Thepolynucleotide of claim 20, comprising a fragment 10 to 150 nucleotidelong that hybridizes to a portion of the PTTG coding region of SEQ. IDNo:
 1. 22. The polynucleotide of claim 19, comprising the PTTG codingregion of SEQ ID NO:
 1. 23. The polynucleotide of claim 19, comprisingthe PTTG coding region of SEQ. ID No:
 3. 24. The polynucleotide of claim23, comprising fragments 10 to 150 nucleotide long of SEQ. ID No:
 3. 25.The polynucleotide of claim 17, which is an RNA.
 26. The polynucleotideof claim 17, which is a DNA.
 27. A vector comprising the polynucleotideof claim
 17. 28. A composition, comprising the vector of claim 27, and acarrier.
 29. A host cell carrying the polynucleotide of claim
 17. 30. Ahost cell carrying the vector of claim
 25. 31. A composition, comprisingthe host cell of claim 29, and a carrier.
 32. An oligonucleotidecomprising at least 15 nucleotides which hybridize under stringentconditions to the nucleic acid comprised in claim
 17. 33. Theoligonucleotide of claim 32, hybridizing under stringent conditions toSEQ ID No:
 1. 34. The oligonucleotide of claim 32, hybridizing understringent conditions to SEQ ID No:
 3. 35. The oligonucleotide of claim32, further comprising a detectable label or marker.
 36. Theoligonucleotide of claim 32, which is an RNA.
 37. The oligonucleotide ofclaim 32, which is a DNA.
 38. An antisense oligonucleotide capable ofhybridizing under stringent conditions to mRNA corresponding to thenucleic acid comprised in claim
 17. 39. A composition, comprising theantisense oligonucleotide of claim 38, and a carrier.
 40. A PTTGdiagnostic kit, comprising at least one oligonucleotide of claim 32, andinstructions for its use in the detection of PTTG encoding DNA.
 41. Amethod of producing a PTTG polypeptide, comprising culturing the hostcells of claim 30, in an expression medium and under effectiveexpression conditions; allowing the PTTG protein to be expressed andaccumulate in the medium; and separating the medium comprising the PTTGprotein from the cells.
 42. The method of claim 41, further comprisingseparating the PTTG protein from the medium and any remainingcomponents.
 43. An isolated anti-PTTG antibody, selectively binding thepolypeptide of claim
 1. 44. The antibody of claim 43, which is amonoclonal antibody.
 45. The antibody of claim 44, which is a polyclonalantibody.
 46. A composition, comprising the antibody of claim 43, and acarrier.
 47. A composition, comprising an amount of the antisenseoligonucleotide according to claim 32, effective to inhibit expressionof a human PTTG gene, and a pharmaceutically acceptable hydrophobiccarrier which passing through a cell membrane.
 48. A transgenicnon-human mammal, carrying at least one non-native polynucleotideexpressing a PTTG protein.
 49. The transgenic non-human mammal of claim48, carrying the polynucleotide of claim
 17. 50. The transgenicnon-human mammal of claim 48, carrying a mutated PTTG encoding nucleicacid, which expresses a non-native PTTG protein.
 51. The transgenicnon-human mammal of claim 48, which is a mouse.
 52. A method ofidentifying mammalian PTTG nucleic acid, comprising contacting thenucleic acid of a mammalian sample with the oligonucleotide of claim 32in labeled form, under stringent hybridization conditions; detecting thepresence of a label in the hybridized nucleic acid of the sample; andidentifying the presence of a mammalian nucleic acid in the presence oflabel in the nucleic acid of the mammalian sample.
 53. A method ofisolating mammalian PTTG nucleic acid, comprising the method of claim52; and separating the labeled hybridized nucleic acid from unlabelednucleic acid from the mammalian sample.
 54. A method of determining thepresence of human PTTG peptide in a sample, comprising contacting ahuman sample with the antibody of claim 43 and allowing the formation ofa complex between the antibody and any PTTG peptide present in thesample; detecting the presence of any antibody-PTTG complex; anddetermining that a human PTTG peptide if any PTTG peptide-antibodycomplex is present in the sample.
 55. The method of claim 54, whereinthe antibody carries a detectable label, and the detecting step iscarried out by detecting the antibody label after sepation of thelabeled complex from unlabeled and labeled antibody.
 56. Single strandPTTG amplification primers\probes, comprising a nucleic acid sequencecorresponding to a nucleic acid selected from the group consisting ofSEQ. ID No: 1, SEQ. ID No: 3, and fragments thereof 10 to 150 nucleotidelong.
 57. A method for detecting a pathological mass associated withPTTG expression, comprising detecting in a subject's cells a transcribedor mutant nucleic acid of a sequence corresponding to the polynucleotideof claim
 17. 58. A PTTG detection kit, comprising the antibody of claim43; and instructions for its use.
 59. The kit of claim 58, furthercomprising a Pituitary-Tumor-Transforming-gene polypeptide selectivelybound by the antibody.